Annonces

Thesis: Identification of structural patterns for antimicrobial peptides recognition at the bacterial cell-­‐wall level

Astrid Walrant (astrid.walrant@sorbonne-­‐universite.fr), Emmanuelle Sachon (emmanuelle.sachon@sorbonne-­‐universite.fr) UMR 7203, Laboratoire des BiomoléculesPôleAnalyse interactions moléculaires et cellulairesSorbonne Université, Paris. ED 406, Chimie moléculaire de Paris Centre (http://www.ed406.upmc.fr/fr/contrats-­doctoraux.html)

 

Membrane-­‐active peptides are a broad class of small peptides with unique membrane interaction properties. Amongst them, cell-­‐penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are of particular interest.

 

Although most AMPs are usually indifferently active on Gram-­‐positive and Gram-­‐negative bacteria, some are more specifically targeted. We recently showed that the AMP DMS-­‐DA6, isolated from a Mexican frog, kills Gram-­‐positive bacteria very specifically (Cardon et al. 2018).

 

The selectivity of AMPs towards bacterial membranes cannot be only the consequence of simple electrostatic interactions between cationic residues on the peptide and anionic lipids. Indeed, both types of bacteria have a negatively charged membrane. Nevertheless, their cell walls differ greatly in terms of surface glycoconjugates. In the case of Gram-­‐positive bacteria, a very thick layer of peptidoglycan (PGN) is found in the most outer part of the bacteria.

 

We recently showed that DMS-­‐DA6 could interact with PGN and that this interaction could potentiate the effect of DMS-­‐DA6 on a lipid bilayer (Cardon et al. 2018). However, so far, we only have indirect evidence for this interaction. Our first objective would thus be to obtain direct evidence of this interaction in vitro. Building on this, our second aim is to identify the structural patterns involved in peptide/PGN and peptide/PGN/lipids using affinity photocrosslinking on reconstituted models. The next step up would then be to evidence interactions on live bacteria.

 

1) In vitro characterisation of DMS-­‐DA6/PGN interaction

 

We plan to study this interaction using various tools that are all available within the host lab, in particular fluorimetry, co-­‐sedimentation assays and isothermal titration calorimetry (ITC). The host team has excellent expertise in this kind of in vitro approach (Walrant et al. 2017).

 

2) Identification of structural recognition patterns by affinity photocrosslinking coupled to mass spectrometry (MS)

 

Affinity photocrosslinking is a powerful technique to study interaction partners, isolate covalent complexes and identify key recognition motifs. Herein, we will develop two complementary approaches to study the interaction partners of DMS-­‐DA6 in either binary systems (peptide/PGN or peptide/membrane) or ternary systems (peptide/PGN/membrane).

 

In the first approach, we will design a DMS-­‐DA6 sequence that will carry a benzophenone (Bzp) photoprobeand a biotin for complex purification and enrichment. We successfully used this type of approach to study the interaction of cell-­‐penetrating peptides (CPPs)and lipids (Jiao et al. 2017, Bechtella et al. submitted). With this approach, our aim is to identify and characterise specific regions interacting with DMS-­‐DA6 on PGN (or PGN analogues) and/or lipids.

 

In the second approach, we will use bi-­‐functionnal lipids, carrying a photoactivable diazirine and analkyne moiety that can be functionalised by click-­‐chemistry (Haberkant et al. 2013). We have recently implemented this approach in our team and are obtaining very promising results with CPPs. This approach is adaptable to any AMP without sequence modification.

 

3 )Photoaffinity crosslinking on live cells

 

Once we have fully characterised the interactions on reconstituted systems, we will move up to partner identification on live bacteria either with the peptide-­‐Bzp or the bi-­‐functionnal lipid strategy. A live cell environment is necessarily more complex, therefore, partner identification in such a context will be challenging.

 

This project should yield valuable information on AMP/PGN/lipids interactions. It will give insighton the general mechanism of membrane permeation by AMPs. It shouldhelp predict the activity of AMPs on bacterial strainsand enable us to rationalise the design of new AMPs with optimised sequences for better bacteria targeting.

 

This project is at the interface of chemistry, biochemistry and biophysics and will involvea large set of techniques: peptide synthesis, mass spectrometry, cell culture, model membranes, calorimetry etc...all available at the host laboratory (LBM).This PhD project will be supervised by Emmanuelle Sachon (HDR) and Astrid Walrant, whose expertise encompassmass spectrometry, affinity photolabelling, membrane active peptides and characterisation of biomolecular interactions.

 

References

 

Bechtella, L., Kirschbaum, C., Clodic G., Matheron, L., Bolbach, G., Sagan, S., Walrant A., Sachon, E., ‘Benzophenone photoreactivity with lipid membranes: simple system, complex information’,

 

Submitted Cardon, S., Sachon, E., Carlier, L., Drujon, T., Walrant, A., Aleman-­‐Navarro, E., Martinez-­‐Osorio, V., Guianvarc’h, D., Sagan, S., Fleury, Y., Marquant, R.,Piesse, C., Rosenstein, Y., Auvynet, C. and Lacombe, C. (2018) ‘Peptidoglycan potentiates the membrane disrupting effect of the carboxyamidated form of DMS-­‐DA6 , a Gram-­‐positive selective antimicrobial peptide isolated from Pachymedusa dacnicolor skin’, PLoS ONE, 13(10), p. e0205727.

 

Haberkant, P., Raijmakers, R., Wildwater, M., Sachsenheimer, T., Brügger, B., Maeda, K., Houweling, M., Gavin, A., Schultz, C., Meer, G. Van, Heck, A. J. R. and Holthuis, J. C. M. (2013) ‘In Vivo Profiling and Visualization of Cellular Protein –Lipid Interactions Using Bifunctional Fatty Acids’, Angewandte Chemie -­‐International Edition, 52(14), pp. 4033–4038. doi: 10.1002/anie.201210178.

 

Jiao, C. Y., Sachon, E., Alves, I. D., Chassaing, G., Bolbach, G. and Sagan, S. (2017)‘Exploiting Benzophenone Photoreactivity To Probe the Phospholipid Environment and Insertion Depth of the Cell-­‐Penetrating Peptide Penetratin in Model Membranes’, Angewandte Chemie -­‐International Edition, 56(28), pp. 8226–8230. doi: 10.1002/anie.201703465.

 

Walrant, A., Cardon, S., Burlina, F. and Sagan, S. (2017) ‘Membrane Crossing and Membranotropic Activity of Cell-­‐Penetrating Peptides: Dangerous Liaisons?’, Accounts of Chemical Research, 50(12), pp. 2968–2975. doi: 10.1021/acs.accounts.7b00455.

Postdoctoral Scientist – Neurobiology
Salary Range £30,914 – £33,755
MRC Laboratory of Molecular Biology, Cambridge, UK

The MRC Laboratory of Molecular Biology is one of the birthplaces of molecular biology and is an internationally renowned centre for fundamental biomedical research.

 

The LMB has a strong track record in training Postdoctoral Fellows who have gone on to become leading Scientists. We also have an excellent record of innovative basic research leading to applications in medicine. Scientists are drawn to the LMB from all over the world, thus creating a lively and international community for the exchange of ideas and technical innovation. Following our move to a new laboratory in 2013, we are able to provide state-of-the-art facilities in which innovative medical research, translation and collaboration can flourish.

 

Applications are invited for a Postdoctoral Scientist position in the group of Dr Ingo Greger to study synaptic glutamate receptor complexes using cryo-electron microscopy (cryo-EM).

 

This project builds on our recent cryo-EM work on heteromeric AMPA-type glutamate receptors (Herguedas et al., Science 2016; Herguedas et al., Science 2019). Candidates should have a PhD and experience with cryo-EM; expertise in membrane protein biochemistry would be greatly advantageous.

 

Successful applicants will be awarded a 3-year Career Development Fellowship/MRC Postdoctoral Training Scheme. The fellowship is a training and development position for a post-doctoral scientist who has recently completed their doctoral studies, or is moving into a new research discipline.

 

Working for UKRI MRC provides a whole host of benefits including a defined benefit pension scheme, excellent holiday entitlement (40 ½ days including bank holidays and privilege days) and Benenden Healthcare membership. You will also have access to employee shopping/travel discounts, on site sport and social facilities, as well as salary sacrifice cycle to work schemes and childcare vouchers.

 

For further information of the research undertaken at the LMB, please visit:
http://www2.mrc-lmb.cam.ac.uk

 

Informal enquires may be made to Dr I. Greger (ig@mrc-lmb.cam.ac.uk).

 

To apply and access full details of the vacancy please visit our job board at https://mrc.tal.net/vx/lang-en-GB/appcentre-1/candidate/postings/1184 . If you are unable to apply online please contact us at recruit@mrc-lmb.cam.ac.uk.

 

Closing date: 29 April 2019
Final appointments will be subject to a pre-employment screening.
The Medical Research Council is an Equal Opportunities Employer who participates in the Disability Confident scheme.

 

Postdoctoral Position: Biomolecular Solid-state NMR

 

The laboratory Membrane Biophysics and NMR at the University of Strasbourg has an opening for a postdoctoral position with experience in using solid-state NMR for the analysis of peptides and proteins. The aim of the project is to reveal the structural determinants that define the highly specific lipid recognition motif of a transmembrane protein and to characterize changes in structure, dynamics, oligomerization and topology of the protein as well as the lipids during recognition. Another ongoing project is the structural investigation of peptide fibers with strong nucleic acid and lentiviral transfection potential.

 

Candidates should have good experience in biomolecular solid-state NMR. Other techniques of the laboratory are solution NMR approaches, various types of biophysical methods, peptide synthesis and/or the biochemical production of proteins. Knowledge in some of these latter techniques are of advantage. S/he should have an interest in working in a highly interdisciplinary, international and collaborative environment. The project and position are funded by a three-year grant from the French National Agency for Research (ANR). The University of Strasbourg chemistry, life sciences and structural biology departments have excellent scientific records, with a multitude of collaborations world-wide.

 

Strasbourg is a very nice city on the French side of the Rhine river, at the border to Germany, with easy access to nearby mountains (Vosges, Black Forrest, Alps). Being in the heart of Europe it takes only short train rides to multiple destinations of scientific and/or touristic interest.

 

Candidates should send their CV, publication list and contact info for three references to:

 

Prof. Burkhard Bechinger,

bechinger@unistra.fr

Web Sites: www-chimie.u-strasbg.fr/~rmnmc

www.icfrc.fr/en/

 

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informations Mentions légales Dernière mise à jour : 17/04/2019


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